Alcohol abuse is a major public health problem leading to premature death, impaired hospital recovery, and dysfunction of multiple organ systems. Muscle wasting is a hallmark of sustained alcohol abuse and the associated proximal muscle weakness represents a prevalent form of skeletal muscle myopathy. During the past funding period, using established rodent models of acute alcohol intoxication and chronic alcohol ingestion, we demonstrated that alcohol impairs not only basal muscle protein synthesis but also the responsiveness of this tissue to nutrient (e.g., leucine) stimulation. This leucine (Leu) resistance results from suppression of mTOR kinase activity, which is manifested as an inhibition of protein synthesis and which appears to be largely Akt/TSC-independent. We also reported that a similar mechanism is operational in muscle cells cultured with alcohol. Our long-term goal is to elucidate the Akt/TSC-independent mechanisms underpinning Leu-induced mTOR activity in skeletal muscle per se and to determine their relative importance in the ability of alcohol to down-regulate this nutritional sensor thereby producing skeletal muscle myopathy. To address the questions implicit in this goal, the proposed research has the following specific aims: (1) Assess the importance of the alcohol-induced change in total and/or phosphorylated DEPTOR (a known negative mTOR-regulatory protein) as a mechanism for the decrease in basal and/or Leu-stimulated muscle protein synthesis; (2) Delineate the mechanism by which alcohol disrupts endosomal trafficking of mTOR complex-1 (mTORC1) and impairs amino acid sensing and protein synthesis in muscle; and (3) Elucidate the extent to which altered MAP4K3 signaling is causally linked to the alcohol-induced decrease in mTOR kinase activity under basal and nutrient-stimulated conditions. Our application exploits a number of innovative approaches made possible by the availability of novel reagents and is supported by provocative preliminary data. It is noteworthy that the proposed in vivo electroporation of lentiviral-delivered shRNA specifically to skeletal muscle permits loss- and gain-of-function experiments to be performed and to assign causality to the observed changes. Furthermore, changes in muscle mass/protein synthesis will be correlated with direct assessment of muscle strength/contractility. These in vivo methods, used in conjunction with an established model of chronic alcohol ingestion in mice and with the availability of novel phospho-specific antibodies, place us in a unique position to rapidl and significantly advance knowledge pertaining to amino acid regulation of mTORC1. Our focus on state-of-the-art in vivo approaches permits us to definitively assign physiological importance to our observations, while complementary in vitro studies will allow us to define cellular mechanisms and to prioritize future work. The expected research outcomes will have a positive impact by contributing fundamental knowledge concerning nutrient regulation at the molecular level and provide seminal mechanistic insights into the clinically significant pathology of alcohol induced muscle disease.